Bottom Line:
Among these proteins, eight novel peroxisome-associated proteins were identified.Although Rho1p has been shown to be tethered to membranes of the secretory pathway, we show that it is specifically recruited to peroxisomes upon their induction in a process dependent on its interaction with the peroxisome membrane protein Pex25p.Rho1p regulates the assembly state of actin on the peroxisome membrane, thereby controlling peroxisome membrane dynamics and biogenesis.

ABSTRACTWe have combined classical subcellular fractionation with large-scale quantitative mass spectrometry to identify proteins that enrich specifically with peroxisomes of Saccharomyces cerevisiae. In two complementary experiments, isotope-coded affinity tags and tandem mass spectrometry were used to quantify the relative enrichment of proteins during the purification of peroxisomes. Mathematical modeling of the data from 306 quantified proteins led to a prioritized list of 70 candidates whose enrichment scores indicated a high likelihood of them being peroxisomal. Among these proteins, eight novel peroxisome-associated proteins were identified. The top novel peroxisomal candidate was the small GTPase Rho1p. Although Rho1p has been shown to be tethered to membranes of the secretory pathway, we show that it is specifically recruited to peroxisomes upon their induction in a process dependent on its interaction with the peroxisome membrane protein Pex25p. Rho1p regulates the assembly state of actin on the peroxisome membrane, thereby controlling peroxisome membrane dynamics and biogenesis.

Mentions:
The positions of peroxisomes and actin patches were analyzed in wild-type and mutant cells, including rho1 cells, containing a genomically encoded Pot1p-GFP chimera to mark peroxisomes. Cells were induced to proliferate peroxisomes, and their actin was labeled with phalloidin-RITC. The relative positions of peroxisomes and actin were determined by double label confocal microscopy (Fig. 9). In wild-type cells, peroxisomes and actin patches showed different localizations, although coincident staining was occasionally observed. However, in rho1-2A cells, peroxisomes and actin patches exhibited virtually exclusive colocalization. Although actin has been proposed to be involved in peroxisome localization within S. cerevisiae (Hoepfner et al., 2001), these data provide evidence for the existence of actin patches on peroxisomes and specifically a role for Rho1p in the organization of actin on this organelle. Remarkably, actin patches were also present on peroxisomes in cells lacking Pex25p, which is required for the proper localization of Rho1p to peroxisomes (Fig. 9). As a comparison, we investigated the dependence of actin localization on Pex11p and Vps1p, which are also implicated in peroxisome division and segregation. In pex11Δ and vps1Δ cells, actin was distributed as in wild-type cells. These data, together with the physical interaction data, suggest that the docking of Rho1p to Pex25p is important for dynamic assembly and disassembly of actin on peroxisomes. Interestingly, vps1Δ rho1 and pex11Δ pex25Δ double mutants also showed accumulation of actin on peroxisomes, which suggests that the majority of actin is reorganized/disassembled before organelle fission and that PEX25 is epistatic to PEX11.

Mentions:
The positions of peroxisomes and actin patches were analyzed in wild-type and mutant cells, including rho1 cells, containing a genomically encoded Pot1p-GFP chimera to mark peroxisomes. Cells were induced to proliferate peroxisomes, and their actin was labeled with phalloidin-RITC. The relative positions of peroxisomes and actin were determined by double label confocal microscopy (Fig. 9). In wild-type cells, peroxisomes and actin patches showed different localizations, although coincident staining was occasionally observed. However, in rho1-2A cells, peroxisomes and actin patches exhibited virtually exclusive colocalization. Although actin has been proposed to be involved in peroxisome localization within S. cerevisiae (Hoepfner et al., 2001), these data provide evidence for the existence of actin patches on peroxisomes and specifically a role for Rho1p in the organization of actin on this organelle. Remarkably, actin patches were also present on peroxisomes in cells lacking Pex25p, which is required for the proper localization of Rho1p to peroxisomes (Fig. 9). As a comparison, we investigated the dependence of actin localization on Pex11p and Vps1p, which are also implicated in peroxisome division and segregation. In pex11Δ and vps1Δ cells, actin was distributed as in wild-type cells. These data, together with the physical interaction data, suggest that the docking of Rho1p to Pex25p is important for dynamic assembly and disassembly of actin on peroxisomes. Interestingly, vps1Δ rho1 and pex11Δ pex25Δ double mutants also showed accumulation of actin on peroxisomes, which suggests that the majority of actin is reorganized/disassembled before organelle fission and that PEX25 is epistatic to PEX11.

Bottom Line:
Among these proteins, eight novel peroxisome-associated proteins were identified.Although Rho1p has been shown to be tethered to membranes of the secretory pathway, we show that it is specifically recruited to peroxisomes upon their induction in a process dependent on its interaction with the peroxisome membrane protein Pex25p.Rho1p regulates the assembly state of actin on the peroxisome membrane, thereby controlling peroxisome membrane dynamics and biogenesis.

ABSTRACTWe have combined classical subcellular fractionation with large-scale quantitative mass spectrometry to identify proteins that enrich specifically with peroxisomes of Saccharomyces cerevisiae. In two complementary experiments, isotope-coded affinity tags and tandem mass spectrometry were used to quantify the relative enrichment of proteins during the purification of peroxisomes. Mathematical modeling of the data from 306 quantified proteins led to a prioritized list of 70 candidates whose enrichment scores indicated a high likelihood of them being peroxisomal. Among these proteins, eight novel peroxisome-associated proteins were identified. The top novel peroxisomal candidate was the small GTPase Rho1p. Although Rho1p has been shown to be tethered to membranes of the secretory pathway, we show that it is specifically recruited to peroxisomes upon their induction in a process dependent on its interaction with the peroxisome membrane protein Pex25p. Rho1p regulates the assembly state of actin on the peroxisome membrane, thereby controlling peroxisome membrane dynamics and biogenesis.